title: Functional and clinicopathological analysis of loss of MKK4 expression in endometrial cancer.
abstract: OBJECTIVE
  In the current study, we investigated the mechanism relating downregulation of mitogen-activated protein kinase kinase-4 (MKK4) expression to the development of endometrial cancer.
  
  
  METHODS
  MKK4 expression in endometrial cancer was assessed by immunohistochemistry using 87 paraffin-embedded tissue specimens, and clinical data was collected via a retrospective chart review. MKK4 gene knockdown using silencing RNA and an MKK4 gene transfection system was used to assess MKK4 function in tissue samples of endometrial cancer.
  
  
  RESULTS
  Lower expression of MKK4 immunointensity was observed in 63.2% (55/87) of the analyzed tumors. High-grade endometrioid adenocarcinoma (G2 and G3) (p = 0.024), postmenopausal status (p = 0.018), and patient age (≥ 60) (p = 0.012) were significantly correlated with lower MKK4 expression. Patients with lower MKK4 expression in endometrial cancer tissues tended to have a shorter overall survival (p = 0.197). Using cell growth and anchorage-independent assays, we determined that both the growth and colony-forming ability of MKK4-transfected HEC1B cells, a line with a low endogenous expression of MKK4, were significantly reduced compared to control vector-transfected cells. Overexpression of the MKK4 gene in HEC1B cells resulted in reduced cell migration activity in a simulated wound healing assay. To confirm that MKK4 expression is related to tumor suppressor function, we used 2 independent but complementary approaches. MKK4 gene knockdown in JHEM1 cells, which overexpressed MKK4, increased proliferation activity. Additionally, the engineered expression of MKK4 in Ishikawa cells, a line with low endogenous MKK4 expression, produced a phenotype similar to that of HEC1B. Similar results were produced in tumor xenografts in nude mice.
  
  
  CONCLUSIONS
  These results indicate that MKK4 acts as a tumor suppressor, and reduced expression of MKK4 may contribute to the development of endometrial cancer.
authors.affiliation: Department of Obstetrics and Gynecology, Shimane University School of Medicine, Izumo, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Ishikawa
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nakayama
authors.forename: Mohammed Tanjimur
authors.initials: MT
authors.lastname: Rahman
authors.forename: Munmun
authors.initials: M
authors.lastname: Rahman
authors.forename: Atsuko
authors.initials: A
authors.lastname: Katagiri
authors.forename: Kouji
authors.initials: K
authors.lastname: Iida
authors.forename: Kohji
authors.initials: K
authors.lastname: Miyazaki
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015153:Blotting, Western; D018269:Carcinoma, Endometrioid / Q000378:metabolism* / Q000473:pathology; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D015536:Down-Regulation; D016889:Endometrial Neoplasms / Q000378:metabolism* / Q000473:pathology; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D007124:Immunoenzyme Techniques; D008207:Lymphatic Metastasis; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015996:Survival Rate; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human